Modification of functional arginine residues in purified bovine testicular hyaluronidase with butane-2, 3-dione.
Purified bovine testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was inactivated by butane-2,3-dione in either borate or Hepes buffer, pH 8.3. The presence of borate enhanced the inactivation process which followed pseudo-first-order kinetics with a calculated second-order rate constant of 13.54M-1 min-1. Using kinetic data it was estimated that the modification of 1 mol arginine per mol enzyme was sufficient for inactivation to occur, whereas amino acid analysis indicated that 4 mol arginine had been modified. The inactivation process was partially prevented by using either competitive inhibitors or substrates of the enzyme, thus indicating that the essential arginine residue is close to the active site of hyaluronidase. A full kinetic analysis of the enzyme with either hyaluronic acid or chondroitin 6-sulphate as substrate showed that the activity of hyaluronidase was uncompetitively activated by either protons or NaCl. The product obtained by reduction of the corboxyl groups of hyaluronic acid to the corresponding alcohol groups was a competitive inhibitor. The possibility that the microenvironment of hyaluronic acid was responsible for the observed kinetic effects of pH and ionic strength was dispelled. It is concluded that these data are compatible with a mechanism that involves anionic interaction between a carboxyl group on the substrate and an arginine residue on the enzyme.
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002074:Butanones / Q000494:pharmacology*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002809:Chondroitin Sulfates / Q000378:metabolism; D003931:Diacetyl / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D009994:Osmolar Concentration; D012965:Sodium Chloride / Q000494:pharmacology; D013737:Testis / Q000201:enzymology*
D002074:Butanones; D012965:Sodium Chloride; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D001120:Arginine; D006821:Hyaluronoglucosaminidase; D003931:Diacetyl